Aquatic Toxicology 130–131 (2013) 97–111 Contents lists available at SciVerse ScienceDirect Aquatic Toxicology jou rnal homepage: www.elsevier.com/locate/aquatox Characterization of cytosolic glutathione peroxidase and phospholipid-hydroperoxide glutathione peroxidase genes in rainbow trout (Oncorhynchus mykiss) and their modulation by in vitro selenium exposure a a b a d c a,∗ D. Pacitti , T. Wang , M.M. Page , S.A.M. Martin , J. Sweetman , J. Feldmann , C.J. Secombes a Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom b Integrative and Environmental Physiology, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom c Trace Element Speciation Laboratory, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, United Kingdom d Alltech Biosciences Centre, Sarney, Summerhill Rd, Dunboyne, Country Meath, Ireland a r t i c l e i n f o a b s t r a c t Article history: Selenium (Se) is an oligonutrient with both essential biological functions and recognized harmful effects. Received 4 July 2012 As the selenocysteine (SeCys) amino acid, selenium is integrated in several Se-containing proteins Received in revised form (selenoproteins), many of which are fundamental for cell homeostasis. Nevertheless, selenium may exert 19 December 2012 toxic effects at levels marginally above those required, mainly through the generation of reactive oxygen Accepted 20 December 2012 species (ROS). The selenium chemical speciation can strongly affect the bioavailability of this metal and its impact on metabolism, dictating the levels that can be beneficial or detrimental towards an organism. Keywords: Glutathione peroxidase (GPxs) is the largest and the most studied selenoprotein family. Cytosolic glu- Selenium tathione peroxidase (cGPx, GPx1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) Sodium selenite Selenocysteine are widely distributed throughout tissues, and play a pivotal role in regulating the oxidative status in the Glutathione peroxidase cell. In this study we have cloned GPx1 and GPx4 genes in rainbow trout (Oncorhynchus mykiss). The con- Salmonids stitutive mRNA expression of these GPx genes was examined in 18 trout tissues and their responsiveness RTL cell line to Se availability was analysed using a rainbow trout liver cell line (RTL). An inorganic (sodium selenite, Na2SeO3) and organic (selenocysteine, Cys-Se-Se-Cys) selenocompound have been used as Se sources. GPx1 activity was also tested to verify the impact of transcript changes on the enzymatic function of these molecules. To understand if the results obtained from the transcript expression analysis were due to Se bioavailability or generation of ROS, the cytoxicity of the two selenocompounds was tested by measuring the impact of Se on cell membrane integrity. Lastly, Se availability was quantified by mass spectropho- tometry to determine the amount of Se in the cell culture media, the Se background due to the foetal calf serum supplement and the contribution from the two selenocompounds used in the treatments. Three isoforms of genes for both GPx1 (GPx1a, 1b1 and 1b2) and GPx4 (GPx4a1, a2 and b) have been identified. The discovery of a third gene encoding for GPx1 and GPx4 hints that salmonids may have the biggest selenoproteome amongst all vertebrates. Transcripts of GPx4 genes were more highly expressed in most tissues examined in vivo (except blood, head kidney and spleen), whereas those of the GPx1 genes were more responsive to selenium exposure in vitro, especially to the organic form. Interestingly, GPx1a was the most sensitive to selenium availability in non stressful conditions, whereas GPx1b1 and GPx1b2 were highly induced by exposure to selenium levels that had some toxic effects on the cells. Although the different concentrations tested of the two selenocompounds modulate GPx1 transcript expression to various degrees, no significant change of GPx1 enzymatic activity was detectable. Our results lead us to conclude that trout GPx1 transcripts expression level may represent a sensitive biomarker for selenium intake, helping to evaluate if selenium concentration and chemical speciation impact on cell homeostasis. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Selenium (Se) is an essential trace element, required as an ∗ Corresponding author at: Scottish Fish Immunology Research Centre, Institute integral part of diverse Se-containing proteins, called seleno- of Biological and Environmental Sciences (IBES), University of Aberdeen, Tillydrone proteins (Burk and Hill, 1993). Through its incorporation into Avenue, Aberdeen, AB24 2TZ, United Kingdom. Tel.: +44 1224 272857; selenoproteins as the selenocysteine (SeCys) amino acid, selenium fax: +44 1224 272872. E-mail address: [email protected] (C.J. Secombes). exerts its biological effects primarily by regulating the antioxidant 0166-445X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2012.12.020 98 D. Pacitti et al. / Aquatic Toxicology 130–131 (2013) 97–111 system and redox enzyme activities within the cell (McKenzie et al., non-specifically into proteins instead of methionine, leading to sig- 2002). Moreover, this trace element is involved in gene transcrip- nificant alterations in protein structure and consequently protein tion and cell signalling cascades, thyroid hormone metabolism, function (Schrauzer, 2000). immune responses and reproduction (Rayman, 2000; Hefnawy and SeCys is the most relevant selenocompound, as the naturally Tórtora-Pérez, 2010). However, Se toxicity can be reached by lev- occurring amino acid (the 21st) that provides the catalytic site for els marginally above those which are required. Among the essential the enzymatic selenoproteins (Stadtman, 1996). SeCys is character- trace elements, Se presents the narrowest range between essential- istically different from the remaining amino acids as it is encoded ity and toxicity, and for this reason it is often difficult to establish by one of the three stop codons (UGA) and is inserted into the [SeCys] which concentrations are beneficial and which become detrimen- selenoproteins by the tRNA , which has specific features that tal towards an organism’s health (NRC, 1980; Foster and Sumar, distinguish it from all other tRNAs (Bock et al., 1991). The UGA 1997). codon is made to encode SeCys by the presence of a cis-acting stem- Diet and water are the two sources of Se. In the aquatic envi- loop structure, designated the SeCys insertion sequence (SECIS) ronment, Se can be absorbed through the gills, gut or epidermis; element, present in the 3 un-translated regions (3 -UTRs) in the however diet is considered the primary source of Se for animals mRNA (Walczak et al., 1996). In addition, other factors are required and the intestine the principal route of assimilation (Dallinger for the incorporation of SeCys into the mature protein, namely et al., 1987; Hamilton, 2004; Janz, 2011). The immediate bioavail- SECIS-binding protein 2 (SBP2) (Copeland et al., 2000), and SeCys- ability of Se depends on its chemical form, which determines the specific elongation factor (EFsec, also called mSelB) (Fagegaltier metabolic and toxic potential (Jonnalagadda and Prasada Rao, 1993; et al., 2000). SECIS elements are present in the 3 un-translated Jackson, 1997; Finley, 2006). Se exists in the environment and in regions (3 -UTRs) of all eukaryotic selenoprotein genes (Berry et al., biological systems as both inorganic and organic forms. The inor- 1991). The general structure of the SECIS element includes two 2− 2− ganic salts, selenite (SeO3 ) and selenate (SeO4 ), contain Se in helices separated by an internal loop, a SECIS core structure located oxidized forms (Se(IV) and Se(VI) respectively), whereas organic at the base of helix 2 and one or two apical loops; its configuration forms contain Se in the reduced state (selenide: Se(−II)) (Thomas can be selenoprotein- and species-specific. The presence of a SECIS et al., 1990; Birringer et al., 2002). Generally, selenocysteine (SeCys) dictates any in-frame UGA codon within the coding region to serve and selenomethionine (SeMet) are the most abundant selenocom- as SeCys when a minimal spacing requirement between UGA and pounds present in the diet (Suzuki, 2005; Dumont et al., 2006). The the SECIS element (51 to 111 nucleotides) is met (Low and Berry, diverse speciation of selenium suggests that the cellular uptake 1996; Fletcher et al., 2001). A set of selenoproteins in an organism of this trace element is likely to occur via multiple membrane is known as the selenoproteome. To date 25 selenoprotein genes transporters (Misra et al., 2012b). Previous studies, focused on the have been indentified in humans (Kryukov et al., 2003). Recent mechanisms of intestinal absorption of selenocompounds in mam- studies on the vertebrate selenoproteome showed that bony fish, 2− mals, suggest that SeO3 absorption occurs by simple diffusion, with 38 selenoprotein genes identified in zebrafish, have a larger 2− whilst SeO4 may be transported by a common transport mech- set of selenoproteins than humans and all terrestrial vertebrates anism with sulphate (Arduser et al., 1985; Wolffram et al., 1986). (Mariotti et al., 2012). SeMet appears to be absorbed by an active transport system shared Glutathione peroxidase (GPxs) is the largest and the most stud- with methionine (McConnell and Cho, 1965; McConnell and